Partikelexpositionen von Zellkulturen: Untersuchungen zur Rolle von Lipidmediatoren bei der inflammatorischen Antwort

Zahlreiche epidemiologische Studien zeigen einen Zusammenhang zwischen der akuten Belastung mit Feinstaub (PM) und dem Auftreten von Lungen- und Herz-Kreislauf-Erkrankungen sowie ansteigenden Sterblichkeitsraten. Die dabei zugrunde liegenden biologischen Wirkungen und die molekularen Mechanismen sind bis heute aber noch weitestgehend ungeklärt. Das Ziel dieser in vitro Studien war es daher sowohl die Mechanismen inflammatorischer und oxidativer Prozesse nach Partikelexposition zu untersuchen als auch deren Verbindung zueinander aufzuklären. Dabei wurden Flugstaubpartikel aus einer Hausmüllverbrennungsanlage (MAF02) als Modell für verbrennungsgenerierte Umweltpartikel eingesetzt. Da Makrophagen, neben Epithelzellen, wichtige Zielzellen der Lunge repräsentieren, wurden zur Untersuchung partikel-induzierter Effekte die murine Makrophagen-Zelllinie RAW264.7, primäre humane Makrophagen aber auch ein aus Makrophagen und Epithelzellen bestehendes Kokultursystem verwendet. Flugaschepartikel induzierten in Makrophagen oxidativen Stress, welcher durch die Generierung von reaktiven Sauerstoffspezies, die Bildung und Freisetzung von 8-Isoprostan, durch eine Erhöhung des intrazellulären Glutathion-Gehaltes, durch eine Akkumulation des Transkriptionsfaktors Nrf2, aber auch durch die Induktion des antioxidativen Hämoxygenase-1 Proteins charakterisiert war. Des Weiteren wurde, als wichtiger Teil der inflammatorischen Antwort, die Freisetzung der Arachidonsäure und deren Metabolisierung zu Prostaglandin E2, ein Vertreter inflammatorischer Mediatoren, nach Partikelbehandlung gezeigt. In Korrelation erfolgte dabei ein Anstieg des Cyclooxygenase-2 Proteins. Interessanterweise konnte in einem realistischeren Kokultursystem mit Makrophagen und Epithelzellen eine synergistisch erhöhte Arachidonsäurefreisetzung im Vergleich zur jeweiligen Monokultur beobachtet werden. In Makrophagen zeigte die Mobilisation der Archidonsäure eine Calcium-Abhängigkeit, was durch den Einsatz der Calciumchelatoren BAPTA/AM und EGTA belegt wurde. Ferner wurde anhand von Inhibitor-Versuchen demonstriert, dass die Flugasche-induzierte Freisetzung der Arachidonsäure vom ERK1/2 und JNK1/2, aber nicht vom p38 MAPK (mitogenaktivierte Proteinkinase) Signalweg abhängig ist. Ausgehend von dieser Beobachtung konnte auf Proteinebene die mit der Aktivierung verbundene Phosphorylierung der ERK1/2 und der JNK1/2 nachgewiesen werden. Des Weiteren bewiesen Studien mitspezifischen Inhibitoren eine Beteiligung der zytosolischen Phospholipase A2, jedoch nicht der sekretorsichen und calcium-unabhängigen Phospholipase A2, an der Flugasche-induzierten Arachidonsäuremobilisation. Die Fähigkeit von MAF02 Partikeln oxidativen Stress zu induzieren und Signalwege, die den Arachidonsäuremetabolismus betreffen, zu aktivieren korrelierte mit der gesteigerten Aufnahme von Partikeln in das Zytosol von Makrophagen. Dabei lagen die Partikel sowohl einzeln als auch in Membraneinschlüssen im Zytosol der Makrophagen vor. Interessanterweise ließ sich die Phosphorylierung von ERK1/2 und JNK1/2 durch den Einsatz des Antioxidanzes, N-Acetylcystein, deutlich reduzieren. Darüber hinaus erfolgte eine N-Acetylcystein-abhängige Inhibierung der Arachidonsäurefreisetzung, woraus erneut geschlossen wird, dass die Aktivierung von ERK1/2 und möglicherweise JNK1/2 an der Arachidonsäurefreisetzung beteiligt sind. Die MAF02-induzierte Induktion der Cyclooxygenase, der Hämoxygenase-1 und die Akkumulation von Nrf2 in der Zelle ließen sich ebenfalls durch NAc hemmen. Zusammenfassend betrachtet belegen diese Studien, dass Flugstaub nach intrazellulärer Aufnahme inflammatorische Prozesse durch die Bildung von reaktiven Sauerstoffspezies initiiert, was zu einer Aktivierung von Signaltransduktionskaskaden und zu einer verstärkten Expression antioxidativer und inflammatorischer Gene führt. Dabei bewirkten zelluläre Interaktionen von Makrophagen und Epithelzellen eine verstärkte Reaktion in Bezug auf die Arachidonsäuremobilisierung nach Flugstaubbelastung. Somit scheint zelluläre Kommunikation entscheidend zur Entwicklung von partikel-induzierten Erkrankungen beizutragen

Regulation of the arachidonic acid mobilization in macrophages by combustion-derived particles

<p>Abstract</p> <p>Background</p> <p>Acute exposure to elevated levels of environmental particulate matter (PM) is associated with increasing morbidity and mortality rates. These adverse health effects, e.g. culminating in respiratory and cardiovascular diseases, have been demonstrated by a multitude of epidemiological studies. However, the underlying mechanisms relevant for toxicity are not completely understood. Especially the role of particle-induced reactive oxygen species (ROS), oxidative stress and inflammatory responses is of particular interest.</p> <p>In this <it>in vitro </it>study we examined the influence of particle-generated ROS on signalling pathways leading to activation of the arachidonic acid (AA) cascade. Incinerator fly ash particles (MAF02) were used as a model for real-life combustion-derived particulate matter. As macrophages, besides epithelial cells, are the major targets of particle actions in the lung murine RAW264.7 macrophages and primary human macrophages were investigated.</p> <p>Results</p> <p>The interaction of fly ash particles with macrophages induced both the generation of ROS and as part of the cellular inflammatory responses a dose- and time-dependent increase of free AA, prostaglandin E₂/thromboxane B₂(PGE₂/TXB₂), and 8-isoprostane, a non-enzymatically formed oxidation product of AA. Additionally, increased phosphorylation of the mitogen-activated protein kinases (MAPK) JNK1/2, p38 and ERK1/2 was observed, the latter of which was shown to be involved in MAF02-generated AA mobilization and phosphorylation of the cytosolic phospolipase A₂. Using specific inhibitors for the different phospolipase A₂isoforms the MAF02-induced AA liberation was shown to be dependent on the cytosolic phospholipase A₂, but not on the secretory and calcium-independent phospholipase A₂. The initiation of the AA pathway due to MAF02 particle exposure was demonstrated to depend on the formation of ROS since the presence of the antioxidant N-acetyl-cysteine (NAC) prevented the MAF02-mediated enhancement of free AA, the subsequent conversion to PGE₂/TXB₂via the induction of COX-2 and the ERK1/2 and JNK1/2 phosphorylation. Finally we showed that the particle-induced formation of ROS, liberation of AA and PGE₂/TXB₂together with the phosphorylation of ERK1/2 and JNK1/2 proteins was decreased after pre-treatment of macrophages with the metal chelator deferoxamine mesylate (DFO).</p> <p>Conclusions</p> <p>These results indicate that one of the primary mechanism initiating inflammatory processes by incinerator fly ash particles seems to be the metal-mediated generation of ROS, which triggers via the MAPK cascade the activation of AA signalling pathway.</p

Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm(2), comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment

Comparing α-Quartz-Induced Cytotoxicity and Interleukin-8 Release in Pulmonary Mono- and Co-Cultures Exposed under Submerged and Air-Liquid Interface Conditions

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm(2) under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations

Contrast of Backscattered Electron SEM Images of Nanoparticles on Substrates with Complex Structure

This study is concerned with backscattered electron scanning electron microscopy (BSE SEM) contrast of complex nanoscaled samples which consist of SiO2 nanoparticles (NPs) deposited on indium-tin-oxide covered bulk SiO2 and glassy carbon substrates. BSE SEM contrast of NPs is studied as function of the primary electron energy and working distance. Contrast inversions are observed which prevent intuitive interpretation of NP contrast in terms of material contrast. Experimental data is quantitatively compared with Monte-Carlo- (MC-) simulations. Quantitative agreement between experimental data and MC-simulations is obtained if the transmission characteristics of the annular semiconductor detector are taken into account. MC-simulations facilitate the understanding of NP contrast inversions and are helpful to derive conditions for optimum material and topography contrast

Silica Nanoparticles Provoke Cell Death Independent of p53 and BAX in Human Colon Cancer Cells

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (–NH2 and –COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53–BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells

Silica nanoparticles are less toxic to human lung cells when deposited at the air-liquid interface compared to conventional submerged exposure

Background: Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air–liquid interface.Results: In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air–liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses.Conclusion: Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method

Boron-rich, cytocompatible block copolymer nanoparticles by polymerization-induced self-assembly

Core-shell nanoparticles (NPs) with a boron-rich core were synthesized by RAFT-mediated polymerization-induced self-assembly using a new methacrylic boronate ester monomer. Under specific conditions, sub-100 nm spherical NPs could be obtained at high conversions by either emulsion or dispersion RAFT polymerization using poly(oligo(ethylene glycol) methacrylate) (POEGMA) dithiobenozate-based chain transfer agents. Phenylboronic acid surface-functionalized NPs were obtained using a telechelic POEGMA. Primary data on biocompatibility is provided and suggests suitability as boron delivery agent for boron neutron capture therapy